Transfection of Lens Cells with Phakinin Mutants

用岩藻蛋白突变体转染晶状体细胞

• 批准号: 6702379
• 负责人: MARK E IRELAND
• 金额: $ 15.1万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-12-01 至 2006-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6702379
• 关键词: animal tissue; cataract; cell differentiation; confocal scanning microscopy; cytoskeleton; gene mutation; immunochemistry; intermediate filaments; lens; lens proteins; molecular assembly /self assembly; protein structure function; tissue /cell culture; transfection

DESCRIPTION (provided by applicant): The long-term objectives of the proposed studies are to determine how the lens-specific beaded filament protein (BFP) cytoskeleton contributes to lenticular clarity. Our overlying hypothesis is that recently identified mutations in the BF subunit phakinin associated with human cataract formation interrupt BF self-assembly or biochemical processing. Specific Aim 1 will test the hypothesis that the in situ biochemical characteristics of BFPs are not altered under in vitro conditions or during the transfection of cultured cells with BF subunit constructs fused to marker protein tags. This will be accomplished with confocal microscopy and immunochemical detection in cultured chicken lens annular pad cells transfected with BF subunit constructs fused to fluorescent proteins (FPs) or the HA epitope. Specific Aim 2 will test the hypothesis that phakinin mutations associated with human cataracts interfere with the polymerization and/or biochemical processing of BFPs during in vitro lens fiber differentiation. This will be accomplished in cultured cells transfected with mutated phakinin subunits fused to fluorescent proteins or antigenic determinants. BF polymerization and biochemical processing will be monitored with confocal microscopy and immunochemical detection of the expressed proteins. The results of these studies will lead to a better understanding of beaded filament function and the etiology of some forms of cataract.

描述（由申请人提供）：拟议研究的长期目标是确定晶状体特异性珠状丝蛋白（BFP）细胞骨架如何有助于晶状体清晰度。我们的首要假设是，最近发现的与人类白内障形成相关的 BF 亚基晶状体蛋白突变会中断 BF 自组装或生化过程。具体目标 1 将检验以下假设：在体外条件下或在用与标记蛋白标签融合的 BF 亚基构建体转染培养细胞的过程中，BFP 的原位生化特性不会改变。这将通过共聚焦显微镜和免疫化学检测在用与荧光蛋白 (FP) 或 HA 表位融合的 BF 亚基构建体转染的培养鸡晶状体环形垫细胞中完成。具体目标 2 将检验以下假设：与人类白内障相关的晶状体蛋白突变会干扰体外晶状体纤维分化过程中 BFP 的聚合和/或生化加工。这将在用与荧光蛋白或抗原决定簇融合的突变的肌红蛋白亚基转染的培养细胞中实现。 BF 聚合和生化过程将通过共聚焦显微镜和表达蛋白质的免疫化学检测进行监测。这些研究的结果将有助于更好地了解珠状丝功能和某些形式白内障的病因学。